Our long-term objective has been to delineate the molecular genetics and pathogenesis of hereditary cardiomyopathies, including arrhythmogenic right ventricular cardiomyopathy (ARVC). ARVC is characterized by fibro-adipocytes replacing cardiac myocytes, which leads to cardiac arrhythmias, sudden cardiac death and heart failure. There is no effective therapy for ARVC, except for heart transplantation. ARVC is primarily caused by mutations in genes encoding desmosome proteins. Desmosomes along with the adherens junctions (AJs) and the gap junctions constitute the intercalated discs (ICDs). While conventionally recognized as cell-cell adhesion structures, ICDs have emerged as major regulators of contact- mediated cell signaling. Accordingly, ICDs are implicated in regulating the Hippo pathway, which plays an important role in cellular differentiation and proliferation. The Preliminary data show extensive molecular remodeling of the ICDs in the human hearts with ARVC. The changes are associated with activation of the Hippo kinase cascade, which by phosphorylating YAP, the effector molecule, suppresses transcription through TEAD. Activation of the Hippo pathway is associated with suppression of the canonical Wnt signaling in the human hearts, which is implicated in the pathogenesis of ARVC. We posit that activation of the Hippo pathway plays a pathogenic role in ARVC. In aim 1, upon further strengthening of the preliminary data, we will delineate the responsible mechanisms for activation of the Hippo pathway utilizing myocyte and mouse models. Preliminary data points to Neurofibromin 1 (NF2), aka Merlin, as an upstream Hippo kinase that is activated at the ICDs in the ARVC models. Through gain- and loss-of-function (GoF and LoF) approaches, the Hippo pathway will be inactivated in myocytes and mouse models and the rescue effects on cardiac structure and function, gene expression and fibro-adipogenesis will be determined. In aim 2, we will delineate the pathogenic role of the canonical Wnt signaling and the mechanisms responsible for its suppression, in the context of active Hippo pathway. GoF and LoF targeting of the canonical Wnt and Hippo molecules will be used to determine the phenotypic effects on myocytes and mouse models. In aim 3, we will identify the cellular origin of fibro-adipocytes in ARVC and determine the pathogenic role of the Hippo and Wnt pathways. The approaches are genetic fate mapping and knock in reporter tracing, the latter for a paracrine effect(s). The candidate cell types, based on the Preliminary data, are fibro-adipocyte progenitors, epicardial cells, pericytes, and myogenic lineage. The paracrine effects of mutant myocytes on the above cells plus resident pre-adipocytes will be tested. The pathogenic role of the Hippo pathway in their differentiation to fibro-adipocytes will be determined through LoF and GoF studies. The proposed studies are expected to provide insights into the molecular pathogenesis of ARVC and facilitate identification of new therapeutic targets for a deadly disease for which there is no effective treatment.